Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering.

ABSTRACT: Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular

SUBMITTER: O'Shea JM 

PROVIDER: S-EPMC9542635 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature